Molecular Fingerprinting by PCR-Denaturing Gradient Gel Electrophoresis Reveals Differences in the Levels of Microbial Diversity for Musty-Earthy Tainted Corks

Abstract

The microbial community structure of cork with marked musty-earthy aromas was analyzed using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown to have a specific microbiota. The fungi Penicillium glabrum and Neurospora spp. were present in all samples and were assumed to make only a small contribution to off-odor development. In contrast, Penicillium islandicum and Penicillium variabile were found almost exclusively in 2,4,6-trichloroanisole (TCA) tainted discs. Conversely, Rhodotorula minuta and Rhodotorula sloofiae were most common in cork stoppers, where only small amounts of TCA were detected. Alpha-and gammaproteobacteria were the most commonly found bacteria in either control or tainted cork stoppers. Specific Pseudomonas and Actinobacteria were detected in stoppers with low amounts of TCA and 2-methoxy-3,5-dimethylpyrazine. These results are discussed in terms of biological degradation of taint compounds by specific microorganisms. Reliable and straightforward microbial identification methods based on a molecular approach provided useful data to determine and evaluate the risk of taint formation in cork.

Bidirectional microbial electron transfer: Switching an acetate oxidizing biofilm to nitrate reducing conditions

“…Bacterial 16S rRNA gene was amplified by PCR using universal primers 357GC (Turner et al, 1999) and 907R (Lane, 1991). PCR products were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) according to the method described by Prat et al (2009). A 35-70% urea-formamide denaturing gradient was used (Supplementary material S1).…”

Estimating Biodiversity of Fungi in Activated Sludge Communities Using Culture-Independent Methods

“…The DGGE profile for each sample was normalised against the marker lanes. The number of bands in each lane was determined and their similarity indices were calculated with the Dice similarity coefficient, a coefficient widely used to compare DGGE profiles in similar fungal biodiversity studies [58,59].…”

Characterization of a novel 2,4,6-trichlorophenol-inducible gene encoding chlorophenol O-methyltransferase from Trichoderma longibrachiatum responsible for the formation of chloroanisoles and detoxification of chlorophenols

“…After a search of putative homologous proteins in bacterial genomes, CMT1 exhibited a low amino acid identity (29% in the best cases) with many O-methyltransferases and putative O-methyltransferase proteins. The capability to produce 2,4,6-TCA at low levels has been reported in some bacterial strains (Allard et al, 1987;Nystrom et al, 1992;Prat et al, 2009), and, therefore, it could be of interest to investigate in the future the putative role of some of these proteins similar to CMT1 in the formation of chloroanisoles in those bacterial strains. Targeted gene disruption is a technique very frequently used to create null mutants of specific genes in fungal strains in order to investigate their roles in metabolism.…”

“…O-methylation of chlorophenols seems to be very usual in filamentous fungi, as it has been documented to occur in many different fungal strains (Cserjesi and Johnson, 1972;Gee and Peel, 1974;Whitfield et al, 1991;Álvarez-Rodríguez et al, 2002;Miki et al, 2005;Maggi et al, 2008). Furthermore, the capability of several bacterial strains to carry out the O-methylation of chlorophenols has been reported (Allard et al, 1987;Neilson et al, 1988;Nystrom et al, 1992;Prat et al, 2009), although the overall contribution of these bacterial strains to the general level of chloroanisoles in the environment seems to be of minor importance. In the fungal strain Trichoderma longibrachiatum, O-methylation of chlorophenols is catalyzed by a novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase, named chlorophenol-O-methyltransferase (CMT1), which has been previously purified and characterized (Coque et al, 2003).…”